Liquid chromatography (LC) is a well-established analytical technique for separating components of a fluidic mixture for subsequent analysis and/or identification, in which a column, microfluidic chip-based channel, or tube is packed with a stationary phase material that typically is a finely divided solid or gel such as small particles with diameter of a few microns. The small particle size provides a large surface area that can be modified with various chemistries creating a stationary phase. A liquid eluent is pumped through the liquid chromatographic column (“LC column”) at a desired flow rate based on the column dimensions and particle size. This liquid eluent is sometimes referred to as the mobile phase. The sample to be analyzed is introduced (e.g., injected) in a small volume into the stream of the mobile phase prior to the LC column. The analytes in the sample are retarded by specific chemical and/or physical interactions with the stationary phase as they traverse the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes or comes out of the end of the column is called the retention time or elution time and can be a reasonably identifying characteristic of a given analyte especially when combined with other analyzing characteristics such as the accurate mass of a given analyte. Or in other words, the analytes interact with the stationary phase based on the partition coefficients for each of the analytes. The partition coefficient is defined as the ratio of the time an analyte spends interacting with the stationary phase to the time spent interacting with the mobile phase. The longer an analyte interacts with the stationary phase, the higher the partition coefficient and the longer the analyte is retained on the LC column. An isocratic flow in LC describes a mobile phase of a constant composition. In contrast to this is the so called “gradient elution”, which is a separation where the mobile phase composition changes during a separation process. For example, a 20-minute gradient starts from 10% MeOH and ends up with 30% MeOH within 20 minutes.
Detection of analytes separated on an LC or nanoLC column can be accomplished by use of a variety of different detectors. Spectroscopic detectors rely on a change in refractive index, ultraviolet and/or visible light absorption, or fluorescence after excitation with a suitable wavelength to detect the separated components. Additionally, the separated components may be passed from the liquid chromatographic column into other types of analytical instruments for further analysis, e.g., liquid chromatography-mass spectrometry (LC/MS or LC-MS) separates compounds chromatographically before they are introduced to the ion source of a mass spectrometer.
The purpose of the LC column is to separate analytes such that a unique response (e.g., a UV absorption peak) for each analyte from a chosen detector can be acquired for a quantitative or qualitative measurement. The ability of a LC column to generate a separation is determined by the dimensions of the column and the particle size supporting the stationary phase. The retention time of an analyte can be adjusted by varying the mobile phase composition and the partition coefficient for an analyte. Increases in chromatographic separation can be achieved via a reduction in the LC column diameter, increasing LC column length and/or a reduction of stationary phase particle dimensions.
Mass spectrometry (“MS” or “mass-spec”) is an analytical technique used to measure the mass-to-charge ratio of gas phase ions. This is achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. A typical mass spectrometer comprises three parts: an ion source, a mass analyzer, and a detector system. The ion source is the part of the mass spectrometer that ionizes the substance under analysis (the analyte). The ions are then transported by magnetic or electric fields to the mass analyzer that separates the ions according to their mass-to-charge ratio (m/z). Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS/MS). The detector records the charge induced or current produced when an ion passes by or hits a surface. A mass spectrum is the result of measuring the signal produced in the detector when scanning m/z ions with a mass analyzer.
Mass spectrometry has rapidly developed as an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. In the second, proteins are enzymatically digested into smaller peptides using an agent such as trypsin or pepsin. The collection of peptide products are then introduced to the mass analyzer. The latter is often referred to as the “bottom-up” approach of protein analysis.
Proteins and/or peptides of interest to biological researchers are usually part of a very complex mixture of other proteins and molecules that co-exist in the biological medium. The high complexity of biological mixtures often makes coupling a separation technique, such as high performance liquid chromatography (HPLC), highly desirable or even required after enzymatic digestion. In addition, HPLC on-line connected to ESI-MS offers the possibility for pre-concentration of dilute samples, desalting and removal of detergents. In many applications, and especially where relatively small volumes of sample are under analysis, improving detection sensitivity can become especially important. Improvement of detection sensitivity using concentration sensitive detectors such as UV/Vis absorbance and ESI mass spectrometers can be achieved by employing HPLC columns with smaller internal diameters (i.d.). For example, increased sensitivity during peptide analysis can result from using nano-LC (e.g., column i.d. of 50-100 μm) and capillary LC (e.g., column i.d. of 320 μm). Flow rate of the mobile phase through such columns is from several nanoliters per minute (nL/min), to several microliters per minute (μL/min), and the mobile phase can be sprayed directly without post-column splitting. The process of electrospray ionization at flow rates on the order of nanoliters (“nL”) per minute has been referred to as “nanoelectrospray ionization” (nanoESI).
Electrospray ionization (ESI) or nanoESI is a commonly applied ionization technique when dealing with biomolecules such as peptides and proteins. The electrospray process creates highly-charged droplets that, under evaporation, create ions representative of the species contained in the solution. An ion-sampling orifice of a mass spectrometer may be used to sample these gas phase ions for mass analysis. When an electric potential or field is applied to the outlet of a conducting needle (often referred to as a sprayer or emitter) relative to an extracting electrode, such as one provided at the ion-sampling orifice of a mass spectrometer, the electric field generated on the needle causes the separation of positively and negatively charged ions in solution and pushes ions of one polarity (e.g., positively charged or negatively charged) to the solution surface. The higher the electric field is the greater the surface charge repulsion force that counteracts the fluid surface tension is. When the repulsion force of the solvated ions exceeds the surface tension of the fluid being electrosprayed, a volume of the fluid is pulled into the shape of a cone, known as a Taylor cone, which extends from the tip of the needle. A liquid jet extends from the tip of the Taylor cone and becomes unstable and generates charged-droplets. These small charged droplets are drawn toward the extracting electrode, e.g., the sampling electrode of a mass spectrometer. The small droplets are highly-charged and solvent evaporation from the droplets results in the excess charge in the droplet residing on the analyte molecules in the electrosprayed fluid. The charged molecules or ions are drawn through the ion-sampling orifice of the mass spectrometer for mass analysis. The potential voltage (“V”) required to initiate an electrospray is dependent on the size of the sprayer, the surface tension of the solution, and the electric field can be on the order of approximately 106 V/m. The physical size of the needle and the fluid surface tension determines the density of electric field lines necessary to initiate electrospray.
In so-called nanoelectrospray, the sample is sprayed from a needle with a tip diameter less than about 5 μm, using a sample flow rate between 5 nL/min and 50 nL/min, for example. Charged droplets with diameters less than 1 micron can be formed at flow rates less than 40 nL/min. These small, highly-charged droplets can provide more efficient ionization of analytes contained within the droplets due to higher surface-to-volume ratios and smaller radii through which analytes need to diffuse to reach the charged surface of the droplets compared to conventional ESI. NanoESI-MS can thus be used for analyzing small amounts of sample with low sample concentrations (e.g., femtomole/microliter). Moreover, with nanoESI, the ion response for analytes contained in a sample solution is proportional to its concentration instead of its total amount. What this means is that if a solution is being sprayed at 200 nL/min or 50 nL/min or 20 nL/min the signal intensity as measured using mass spectrometry would be the same. Thus reducing a flow rate by a factor of 5 roughly increases mass spectrometry scans to be acquired for the same amount of sample by a factor of 5. As a result, signal averaging from the increased number of scans improves signal-to-noise ratios and ion statistics which enable multiple MS/MS experiments on the analytes and high accuracy in identifying analytes.
Tandem mass spectrometry (MS/MS) is a popular experimental method for identifying biomolecules such as proteins. Tandem MS involves multiple steps of mass selection or analysis, usually separated by some form of fragmentation. A tandem mass spectrometer is capable of multiple stages of mass spectrometry. For example, one mass analyzer can isolate one peptide from many others entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then characterizes the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD).
Characterization of total digests (peptides) of complex protein mixtures using HPLC/MS is also called shotgun proteomics. Shotgun analysis involves direct digestion of protein mixtures to complex peptide mixtures, followed by the automated identification of the peptides by liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). The development of shotgun proteomics today couples nano-LC for peptide analysis, automated MS to MS/MS data acquisition software and hardware on modern tandem mass spectrometers, along with data searching software of modern search engines. Modern mass spectrometers with data dependent scanning software are capable of acquiring MS and MS/MS data from many hundreds of peptides per hour.
The principle of shotgun analysis is to first “spread out” peptides in complex mixtures by multidimensional chromatographic separation. Tandem MS instruments then acquire peptide MS/MS spectra, which encode the peptide sequences. This acquisition can be done in a data-dependent way, whereby the instrument relies on a preliminary scan, performed as the peptides enter the instrument, to select peptides for fragmentation and generation of MS/MS spectra.